The principal resorptive cells of bone, the osteoclasts, attach to bone through integrin-dependent adhesion structures known as podosomes. Prior to initiation of bone resorption, these podosomes form putative precursors of the sealing zone, and are similar to focal adhesions. Several protein-tyrosine kinases and phosphorylated proteins exist within the podosomal signaling complex. Integrin activation results in an increased tyrosine-phosphorylation of the nonreceptor protein tyrosine kinase (Pyk2) and induces its association with other signaling molecules. The characterization of the wide array of signaling molecules contained within the osteoclast (OC) podosomal complex is largely incomplete. The aim of this grant application is to characterize the role of Leupaxin (LPXN), a protein that we have identified as a novel component of the OC podosomal signaling complex. LPXN is a cytoplasmic tyrosine-phosphorylated protein that shares homology with the focal adhesion protein, paxillin. We have found that LPXN is associated with both the protein-tyrosine kinases, Pyk2 and FAK, and with the protein-tyrosine phosphatase, (PTP)-PEST, at the podosomal complex, suggesting regulation by phosphorylation / dephosphorylation mechanisms. LPXN was also found to associate with the ARF-GTPase paxillin kinase linker, p95PKL, the p21GTPase-activated kinase, PAK, and the structural focal adhesion actin-binding protein, actopaxin. Both tumor necrosis factor-alpha, and an extracellular matrix protein, such as osteopontin, were found to stimulate tyrosine-phosphorylation of LPXN. Overexpression of LPXN evoked numerous cytoplasmic projections at the leading edge of the cell, resembling a motile phenotype. Finally, in vitro inhibition of LPXN expression in the OC led to a decrease in resorptive capacity. We hypothesize that LPXN functions as an adaptor protein, and is a critical nucleating component of the OC podosomal signaling complex. This study will determine the role of LPXN in the regulation of OC activity, regulation of LPXN by protein tyrosine kinases, and functional significance of interaction between LPXN and PTP-PEST. We propose that LPXN, as a podosomal scaffold protein, plays a role in OC motility and resorption by regulating adhesion and turnover of the podosomal complex. The identification of LPXN as a new component of the OC adhesion zone adds a new complexity to their regulation, and may provide a pharmacological target in bone resorption.